Test methods (assays) for the detection of analytes in samples are widely known in the art. Particular examples of assays are those whereby the analyte to be detected has a specific color-, IR-, or UV-spectrum and can thus be detected by means of a reader comprising a photoelectric cell, a scanner, any type of camera and of course visually. If the spectral characteristics of the analyte in question are not sufficient for adequate detection it is often possible to use indirect methods. For instance, methods are available whereby the presence of the analyte triggers a secondary process, which then results in the formation of a product or event having a specific spectral characteristic. This may be a colored product, but can also be a change in pH, redox-potential, temperature and the like, which in turn triggers an indicator molecule to change color-, IR-, redox- or UV-spectrum. Many types of secondary processes are employed in the art for various applications.
One example is a chemical reaction of the analyte with a fluorescent or colored molecule resulting in a product without fluorescence or color or with a different fluorescence or color, or the other way around.
Another example is inhibition of a (bio) chemical process by the analyte whereby the presence of the analyte is indirectly measured as a result of the absence (or presence) of the effect of that (bio) chemical process. The latter may be illustrated by, for instance, microbiological assays for the detection of analytes, particularly residues of antibiotics and chemotherapeutics, in fluids such as milk, meat juice, serum, urine, (waste) water and the like. Examples of such assays have been described in CA 2056581, DE 3613794, EP 0005891, EP 0285792, EP 0611001, GB A 1467439 and U.S. Pat. No. 4,946,777. These descriptions all deal with ready to use assays that make use of an assay organism and will give a result by the change indicated by an indicator molecule, for instance a change of color of a pH- and/or redox-indicator, added to the assay. A change in the indicator indicates the presence of a growing assay organism. The principle is that when an analyte is present in a fluid in a concentration sufficient to inhibit growth of the assay organism, the color of the indicator will stay the same. In contrast, when no inhibition occurs, growth of the assay organism is accompanied by the formation of acid or reduced metabolites or other phenomena that will induce an indicator signal. The known assays mentioned above include an assay medium, such as an agar medium, inoculated with a suitable assay organism, preferably a strain of Bacillus, Escherichia or Streptococcus, and a pH indicator and/or a redox indicator. The suitable assay organism and the indicator, and optional buffers, nutrients, surfactants and the like, are introduced into a gel or simply kept in solution. Normally conditions are chosen in such a way that the assay organisms stay alive but cannot multiply because of lack of an essential growth requirement (this may be a nutrient, a specific pH- or temperature value or any other essential parameter).
Assays that require a more or less stringent temperature regime for the results to be generated reliably and accurately form a special class of methods amongst the ones mentioned above. These types of methods require incubation equipment in order to maintain the assay at a predetermined temperature or temperature profile for a given period of time. Usually the result of such an assay is determined afterwards by using a reader equipped to detect the event that indicates the presence of the analyte. An example of such an assay is described in WO 03/033728 dealing with a method for detecting the presence of an analyte (such as a β-lactam) in a sample (such as food products, e.g. meat, milk, or body fluids, e.g. blood, urine) following incubation at 64° C. by determining color values of the sample, associated with the L*a*b color model, using a standard scanner coupled to a computer. The latter reading technology was also disclosed in EP 953 149.
A severe disadvantage of these prior art methods is that it is only possible to perform reading operations after incubation. As a result of this, diagnostic methods such as microbiological assays for the detection of antimicrobial residues only give a positive or negative result (i.e. merely indicate whether or not the concentration of analyte is above or below a certain threshold value). In case of a positive result obtained with these so-called screening assays, the sample has to be examined further for confirmation using a second diagnostic method. Mostly, such confirmation methods, e.g. HPLC or mass spectrometry, are extremely expensive and it takes a long time before the results are known. Thus, reading assay results during the screening assay itself would be advantageous, as this would give access to more detailed information that could improve on parameters such as assay duration, type of analyte or concentration of analyte.
Nevertheless, reading of information during an assay and simultaneous incubation of such an assay is known in methods based on the presence of a light source on one end of the assay and collecting light signals on the other end of the assay, an example of which are the well-known ELISA-readers. This principle has, unfortunately, three major disadvantages. Firstly, in assays wherein the sample is placed on a substrate and the required reaction takes place within the substrate, the nature and amount of the sample will disturb the measurement when light or another type of radiation travels through both sample and substrate. Secondly, the equipment usually employed for such measurements is mostly dedicated and designed for trained personnel and to be used in a laboratory environment. Thirdly, the prior art principles are designed to measure one particular wavelength only. There is thus a need for equipment and methods that do not suffer from these drawbacks.